Easysep mouse pan ilc enrichment kit

To isolate single cells from lungs, lung tissues were digested with RPMI 1640 media (Welgene, Gyeongsangbuk-do, South Korea) supplemented with collagenase type IV (1 mg/ml; Worthington Biochemical Corp, NJ, USA) and DNase I (0.5 mg/ml; Sigma-Aldrich, MO, USA) for 90 min at 37°C. The digested tissues were filtered with 40-μm cell strainer and the red blood cells (RBCs) were lysed with RBC lysis buffer (BioLegend, CA, USA). To isolate the ILC2s from the lungs, mice were treated intratracheally with 500 ng of IL-33 (BioLegend) for 3 consecutive days and, after 7 days, the lungs were digested as described above. The ILC2s were then isolated with the EasySep Mouse Pan-ILC Enrichment Kit (STEMCELL Technologies Inc., Vancouver, Canada).

ILC2s were isolated from the lungs of naïve mice by using EasySep™ Mouse Pan-ILC Enrichment Kit (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer’s instructions. pDCs were isolated from bone marrows of naïve mice using EasySep™ Mouse Plasmacytoid DC Isolation Kit (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer’s instructions. Thus, bone marrow was flushed out with PBS and filtered with a 40-μm strainer. Red blood cells were lysed by RBC Lysis Buffer (BioLegend) and the cells were washed with PBS. Sorted 1.0 × 10⁵ ILC2s were cultured with 1.0 × 10⁵ pDCs in DMEM media at 37°C, 5% CO₂. Recombinant IL-2 (10 ng/mL, BioLegend), IL-7 (10 ng/mL, BioLegend), IL-33 (10 ng/mL, BioLegend), and ssRNA40 (1 μg/mL, Invitrogen) were simultaneously added into the co-culture plate. In some groups, anti-mouse interferon-alpha receptor subunit 1 (IFNAR-1) (20 μg/mL, BioXCell, Lebanon, NH, USA) was added in co-culture. After 48 hours, the cells were analyzed by flow cytometry, and the supernatant was analyzed by ELISA.